The field of embryo transfer is growing in each animal sector in which multiple offspring are desirable. There were over 130,000 donor cattle superovulated worldwide in 2006 and the number of transferred embryos increased by 10% to over 670,000 (IETS Newsletter December 2007). In the United States, there were an estimated 52,000 donors superovulated in 2006 (AETA Annual Report 2006). The current superovulation protocols all require multiple injections of Follicle Stimulating Hormone (FSH) twice daily over the course of at least 4 days. The FSH currently used is animal derived, impure and has the propensity to be infectious. The invention described herein is a long-acting FSH analog that is effective in causing superovulation with a single injection. Furthermore, this FSH analog is highly purified and free of infectious vectors and other contaminants.
There are over nine million dairy cows in the United States, over one million in Canada and over fifty million worldwide. The dairy industry is extremely competitive and the ability of a dairy to increase the efficiency of breeding and to maintain pregnancies post insemination is critical to the profitability of the producer. It is estimated that the cost of a non-pregnant cow is about five dollars per day. It is further estimated that current inseminations result in approximately twenty to thirty-five percent pregnant cows at day 45 and of those cows ninety to ninety-five percent deliver calves at the end of the 283-day gestation period. However, reproductive efficiency in dairy cattle has been declining steadily over a prolonged period of time. The magnitude and the consistency of this trend are of great importance to the dairy industry and amount to a steady decline of approximately one percent in first service conception rates per year for the last ten years. The impact of this change in productivity has not been readily apparent, because individual cow milk production has increased by twenty percent over the same period. In the long run, the dairy industry cannot afford to continue the current rate of declining reproductive performance.
Declining reproductive efficiency of dairy cattle has been observed throughout the United States, and other parts of the world where milk production has been increasing (Lucy, M. C. (2001) “Reproductive loss in high-producing dairy cattle: Where will it end?,” J. Dairy Sci., 84:1277-1293; Roche et al. (2000) “Reproductive management of postpartum cows,” Anim. Reprod. Sci., 60-61:703-712; Royal et al. (2000) “Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility,” Anim. Sci., 70:487-502; and Macmillan et al. (1996) “The effects of lactation on the fertility of dairy cows” Aust. Vet. J, 73:141-147). Numerous features may negatively influence fertility in dairy cows, including negative energy balance and disease events such as retained placenta, ketosis, cystic ovary, and mastitis (Lucy 2001, supra; and Staples et al. (1990) “Relationship between ovarian activity and energy status during the early postpartum period of high producing dairy cows,” J. Dairy Sci., 73:938-947). Furthermore, a prominent trend in the U.S. dairy industry is decreased number of dairy farms, steadily increasing herd size, and movement of dairy production to the western states (USDA National Agricultural Statistics Service, http//www.usda.gov/nass). Larger herd size may contribute to decreased reproductive performance because of the associated changes in the dairy labor force and cow management, resulting in poorly trained or over tasked workers identifying estrus behavior, performing artificial insemination, conducting estrus synchronization programs, and identifying and treating sick cows (Lucy 2001, supra). Heat stress, which occurs throughout much of the year in western and southwestern US dairy herds, has significant negative impact on cattle fertility (Wolfenson et al. (2000) “Impaired reproduction in heat-stressed cattle: basic and applied aspects,” Anim. Reprod. Sci., 60-61:535-547).
The primary revenue source in the dairy industry is milk production. Progress in genetics and management of dairy cows has led to remarkable increases in milk production throughout the last several decades, with a twenty percent increase in per-cow production in the last ten years alone (USDA National Agricultural Statistics Service, http//www.usda.gov/nass). In order to maintain high herd productivity, however, cows must become pregnant and deliver a calf so that the lactation cycle is renewed. Additionally, sufficient numbers of heifers must be produced to replace older cows. Therefore, the future productivity of the dairy industry is very dependent on the maintenance of fertility and reproduction.
The ability to increase reproductive performance in horses, cattle or other ungulates would have a significant economic benefit to owners. This can be achieved through increasing fertility as well as improving pregnancy maintenance throughout the gestation period to prevent pregnancy losses. Recent studies with ultrasonic pregnancy detection demonstrate embryonic losses in cattle of at least 20% between 28 and 60 days of pregnancy (Pursley et al. (1998) “Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows,” J. Dairy Sci., 81:2139-2144; and Vasconcelos et al. (1997) “Pregnancy rate, pregnancy loss, and response to heat stress after AI at 2 different times from ovulation in dairy cows” Biol. Reprod., 56 (Supp. 1):140). There are likely even higher losses prior to 28 days that are undetected by ultrasound examination (Lucy 2001, supra). Data suggest that modern dairy cows fail to establish pregnancy because of suboptimal uterine environment (Gustafsson, H. and K. Larsson (1985) “Embryonic mortality in heifers after artificial insemination and embryo transfer: differences between virgin and repeat breeder heifers,” Res. Vet. Sci., 39:271-274). Although there are numerous possible factors that could be responsible for embryonic losses, one potential cause is low blood progesterone concentration.
Currently, several hormone therapies are used to increase fertility or to maintain pregnancy. Thatcher et al. (2001 Theriogenology 55:75-89) describes the effects of hormonal treatments on the reproductive performance of cattle. Hormonal treatments include administration of bovine somatotrophin (bST) and human chorionic gonadotropin (hCG). D'Occhio et al. (2000 Anim. Reprod. Sci. 60-61:433-442) describes various strategies for beef cattle management using gonadotropin releasing hormone (GnRH) agonist implants. De Rensis et al. (2002 Theriogenology 58(9):1675-1687) describes the effect on dairy cows of administering GnRH or hCG before artificial insemination. Martinez et al. (1999 Anim. Reprod. Sci. 57:23-33) describes the ability of porcine luteinizing hormone (LH) and GnRH to induce follicular wave emergence in beef heifers on Days 3, 6, and 9 of the estrus cycle, after ovulation (Day 0), without insemination. Santos et al. (2001 J. Animal Science 79:2881-2894) describes the effect on reproductive performance of intramuscular administration of 3,300 IU of hCG to high-producing dairy cows on Day 5 after artificial insemination. Lee et al. (1983 Am. J. Vet. Res. 44(11):2160-2163) describes the effect on dairy cows of administering GnRH at the time of artificial insemination. U.S. Pat. No. 5,792,785 (issued Aug. 11, 1998) and U.S. Pat. No. 6,403,631 (issued Jun. 11, 2002) describe methods and compositions for administering melatonin before and after insemination to enhance pregnancy success in an animal. Chagas e Silva et al. (2002 Theriogenology 58(1):51-59) describes plasma progesterone profiles following embryo transfer in dairy cattle. Weems et al. (1998 Prostaglandins and other Lipid Mediators) describes the effects of hormones on the secretion of progesterone by corpora lutea (CL) from non-pregnant and pregnant cows. U.S. Pat. No. 4,780,451 (issued Oct. 25, 1988) describes compositions and methods using LH and follicle stimulating hormone (FSH) to produce superovulation in cattle. Farin et al. (1988 Biol. Reprod. 38:413-421) describes the effect on ovine luteal weight of intravenous administration of 300 IU of hCG on Days 5 and 7.5 of the estrus cycle, without insemination. Hoyer and Niswender (1985 Can. J. Physiol. Pharmacol. 63(3):240-248) describe the regulation of steroidogenesis in ovine luteal cells. Juengel and Niswender (1999 J. Reprod. Fertil. Suppl. 54:193-205) describe the molecular regulation of luteal progesterone in domestic ruminants. U.S. Pat. No. 5,589,457 (issued Dec. 31, 1996) describes methods for synchronizing ovulation in cattle using GnRH, LH, and/or hCG and PGF2α.
Many of these treatments use hormones or hormone analogs from the glycoprotein hormone family, which consists of the pituitary proteins luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG). The gonadotropins, which include CG, FSH and LH, are essential for reproductive function. They are heterodimers composed of two non-covalently associated α and β subunits. Both subunits are glycosylated, containing asparagine (N)-linked oligosaccharides and, in the case of the CGβ subunit, O-linked carbohydrates are also present in a cluster of amino acids at the C-terminus. The individual human β subunits are encoded by separate genes, and the LHβ and CGβ proteins are structurally and functionally similar; having more than 80% amino acid identity (Pierce J G, Parsons (1981) “Glycoprotein hormones: structure and function,” Biochem. 50:465-495). Within a species, the α subunit amino acid sequence is common to all four hormones (Pierce J G, Parsons (1981) Biochem. 50:465-495).
In order to use gonadotropins to improve reproduction efficiency in animals, the availability of purified proteins is essential. Currently, the sources for gonadotropins are serum and whole pituitary extracts. To obtain sufficient quantities of these native hormones for such work is expensive and difficult. Pituitary extracts can be effective reproductive therapeutics but contain contaminants and may vary in their amounts of LH and FSH. Preparations of pure pituitary gonadotropins without cross-contamination are not readily available. Given the problem of animal-to-animal variation of native gonadotropins and the charge heterogeneity in the N-linked carbohydrates, the ability to generate the corresponding recombinant proteins will yield gonadotropins of a more homogeneous composition that can be standardized with respect to mass and bioactivity. Such proteins will be critical for calibrating clinical laboratory assays and for breeding management, such as shortening the time to ovulation in transitional and cycling mares for natural breeding and artificial insemination. The use of recombinant forms, as opposed to hormones extracted from serum and pituitary tissue, would avoid the co-contamination of pathogens and agents with a propensity to cause prion related diseases.
Thus, what are needed are active recombinant gonadotropin analogs, particularly FSH analogs, and methods of using such analogs to improve reproduction of cattle and other animals.